Characterization of phosphorylation dependent antibodies to study the phosphorylation status of the Tau protein

The microtubtile-associated protein Tau (T) regulates the assembly and disassembly of neuronal rnicrotubules. In Alzheimer's disease (AD), T becomes hyperphosphorylated and aggregates to form paired helical filaments (PHF). As the phosphorylation status of normal and biopsy-derived T versus PHF-tau is still unclear, there is need for antibodies recognizing a distinct phosphorylation pattern without cross-reactivity. Thus, we studied seven phosphorylation-dependent antibodies directed towards phosphoserine and phosphothreonine residues in positions 212, 214, 217, 231, 396, 400, and 404 of human T (numbering according to the longest splicing-form with 441 residues). In an immunosorbent assay only one antibody showed a significant cross-reactivity towards the unmodified sequence. All other antibodies recognized only the phosphorylated sequences at lower peptide concentrations typically applied in immunosorbent assays. However, the binding of antibodies directed towards Thr212, Thr217, and Ser400 were reduced when the nearby Ser214, Thr212 or Ser396, respectively, were simultaneously phosphorylated. The phosphate specificity was confirmed on the protein level using bovine tau in its native phosphorylation status as well as T dephosphorylated by phosphatases. Immunoblot analyses after two-dimensional gel electrophoreses also indicated that the pAbs recognized specifically the phosphorylated T-versions.